The present invention relates to a molecularly imprinted polymer, to a method for preparing said molecularly imprinted polymer, and to the use of said molecularly imprinted polymer.
In the fields of medical, dietary, environmental and chemical sciences there is an increasing need for the selective separation of specific substances in complex mixtures of related substances. The end goal can be the preparative isolation of a certain compound or compounds or measurements of their concentration. Molecularly imprinted polymers (MIPs) often exhibit a high selectivity towards their substrate in analogy with the antibody-antigen complementarity. (1, 2) The technique shows promise in chiral separations of for example amino acid derivatives, peptides, phosphonates, aminoalcohols and beta-blocking compounds, affinity chromatography of nucleotides and the DNA-bases as well as substitute for antibodies in immunoassays for commercial drugs. Molecular imprinting (MI) consists of the following key steps: (1) Functional monomers are allowed to interact reversibly with a template molecule in solution. (2) The hereby formed template assemblies are copolymerised with a cross-linking monomer resulting in a cross-linked network polymer. (3) The template is displaced and the resulting MIP material can be used for selective molecular recognition of the corresponding compound. If the MIP material is crushed and sieved it can be-packed in a chromatographic column and used for chromatographic separation of the template from structurally related analogs. Analytical as well as preparative applications are here possible. Preparative applications can be separation of a compound from a complex mixture of structurally related compounds and isolation of the compound. This can be through an affinity chromatographic procedure where pH, ion strength or solvent gradients can be used in order to control the strength of interaction with the stationary phase The separation can target enantiomers or diastereomers in a mixture of enantiomers or diastereomers of one or many compounds. Analytical applications can in addition to the above mentioned separations be: competetitive binding assays, chemical sensors or selective sample enrichments.
Currently the most widely applied technique to generate molecularly imprinted binding sites is represented by the non-covalent route developed by the group of Mosbach(3). This makes use of non-covalent self-assembly of the template with functional monomers prior to polymerisation, free radical polymerisation with a cross-linking monomer and then template extraction followed by rebinding by non-covalent interactions. Although the preparation of a MIP by this method is technically simple it relies on the success of stabilisation of the relatively weak interactions between the template and the functional monomers. Stable monomer-template assemblies will in turn lead to a larger concentration of high affinity binding sites in the resulting polymer. The materials can be synthesized in any standard equipped laboratory in a relatively short time and some of the MIPs exhibit binding affinities and selectivities in the order of those exhibited by antibodies towards their antigens. Most MIPs are synthesized by free radical polymerisation of functional monounsaturated (vinylic, acrylic, methacrylic) monomers and an excess of cross-linking di- or triunsaturated (vinylic, acrylic, methacrylic) monomers resulting in porous organic network materials. These polymerisations have the advantage of being relatively robust allowing polymers to be prepared in high yield using different solvents (aqueous or organic) and at different temperatures (4). This is necessary in view of the varying solubilities of the template molecules.
The most successful non-covalent imprinting systems are based on commodity acrylic or methacrylic monomers, such as methacrylic acid (MAA), cross-linked with ethyleneglycol dimethacrylate (EDMA). Initially, derivatives of amino acid enantiomers were used as templates for the preparation of imprinted stationary phases for chiral separations (MICSPs) but this system has proven generally applicable to the imprinting of templates allowing hydrogen bonding or electrostatic interactions to develop with MAA.(5, 6) The procedure applied to the imprinting with L-phenylalanine anilide (L-PA) is outlined in FIG. 1. In the first step, the template (L-PA), the functional monomer (MAA) and the cross-linking monomer (EDMA) are dissolved in a poorly hydrogen bonding solvent (diluent) of low to medium polarity. The free radical polymerisation is then initiated with an azo initiator, commonly azo-N,Nxe2x80x2-bis-isobutyronitrile (AIBN) either by photochemical homolysis below room temperature(6, 7) or thermochemically at 60xc2x0 C. or higher(5). Lower thermochemical initiation temperatures down to 40xc2x0 C. or 30xc2x0 C. may be obtained using azo-N,Nxe2x80x2-bis-divaleronitrile (ABDV) and V70 resp. instead of AIBN as initiator (see). (7, 8) In the final step, the resultant polymer is crushed by mortar and pestle or in a ball mill, extracted by a Soxhlet apparatus, and sieved to a particle size suitable for chromatographic (25-38 xcexcm) or batch (150-250 xcexcm) applications. (6) The polymers are then evaluated as stationary phases in chromatography by comparing the retention time or capacity factor (kxe2x80x2) (9) of the template with that of structurally related analogs.
As appears from above MIPs have sofar been prepared in the form of continuous blocks that need to be crushed and sieved before use. This results in a low yield of irregular particles, a high consumption of template and a material exhibiting low chromatographic efficiency. There is therefore a need for MI-materials that can be prepared in high yield in the form of regularly shaped particles with low size dispersity and a controlled porosity. These are expected to be superior in terms of mass transfer characteristics and sample load capacity compared to the materials obtained from the monolithic approach.
Such MIPs have been previously prepared through suspension(10, 11)xe2x80x94polymerisation techniques, dispersion polymerisation(12) or precipitation polymerisation(13). This resulted in spherical particles of a narrow size distribution. These procedures have the limitation of being very sensitive to small changes in the manufacturing conditions and the type of solvents and polymerisation conditions that can be applied. Thus the procedures need careful optimization for each new template target which significantly reduces the usefulness of this route. Moreover conditions leading to low dispersity spherical particles may not be compatible with conditions leading to high selectivity and affinity for the template target.
An alternative to this procedure is the coating of preformed support materials. (14-16) MIPs have been prepared as grafted coatings on oxide supports(14, 16) on organic polymer supports(15) and on the walls of fused silica capillaries(17-19). The former technique allows the use of the wide variety of oxide support materials available with different sizes and porosities. Grafting techniques to prepare organic polymer coatings are expected to be generally applicable to molecular imprinting since the structure of the underlying support is already fixed. Thus compared to the large number of factors influencing the end result in suspension or precipitation type polymerisations a smaller number of factors is likely to influence the end result in the preparation of the imprinted coatings. This will make the grafted coatings techniques less sensitive to changes in conditions offering a more robust method. These types of coating techniqes are furthermore applicable to modify surfaces of monolithic type supports or microchips prepared by lithographic techniques. The oxide based materials are rigid porous supports with a limited inner pare volume. An alternative support that could potentially carry more grafted imprinted polymer per unit weight and thus allow a higher density of imprinted sites would be to make use of swellable organic resins. In this context Merrifield resins containing grafted initiator or monomer could be used.
So far most imprinted coatings have been prepared by grafting polymers to the various surfaces. Thus she surface contains prior to polymerisation polymerizable double bonds that can add to the growing polymer chains in solution linking them to the surface. The problem with this technique is the presence of initiator in solution requiring the monomer mixture to be applied as a liquid thin film on the surface prior to polymerization. Thus the exact amount of monomers that will coat the available surface with an up to ca 100 xc3x85 thick liquid film is dissolved together with initiator in an excess of solvent. Thereafter the modified support is added and the solvent evaporated to leave the monomer film and initiator on the surface. Polymerisation is then carried out usually at elevated temperatures. With this procedure the thickness of the polymer layer is difficult to control and capillary forces upon evaporation of solvent may cause incomplete wetting of the surface. Moreover a continuous method of synthesising the particles is difficult to envisage with this method.
A considerable improvement in this regard would be to confine the initiator radicals to the support surface. (FIG. 2). (20, 21) In absence of chain transfer this would lead to chain growth occuring only from the surface of the support with no polymerisation occuring in solution. For molecular imprinting this would have important consequences. For instance the polymerisation can be carried out on the surface of initiator modified support particles suspended in a mixture of the monomers and solvent. This would allow polymerisation in a simple tank reactor by either thermal or photochemical initiation. The latter technique would allow the particles to be modified during the sedimentation possibly leading to a continuous method for preparing the imprinted composite particles (FIG. 3). Polymerisation would here only occur on the particle surface leaving the solution containing the monomers unreacted. The monomer solution can thus be reused for the coating of several batches of particles. The problem of confining polymer chain growth to the support surface and suppress it in solution can be solved by attaching the radical initiator so that the radical formed upon bond homolysis remains bound to the surface. Alternatively the radical formed that is not attached to the surface should undergo rapid reaction to give an unreactive species. It should be possible to prepare the grafted coatings using monomers such as those based on styren/divinylbenzene, methacrylates, acrylates, acrylamides and in the presence of one or more template molecules.
Thus, the present invention relates to a molecularly imprinted polymer obtainable by polymerising a composition comprising at least one monomer, and a template, on a support in a polymerisation medium with a free radical initiator, whereafter the template is removed from the molecularly imprinted polymer obtained, said polymerisation being confined to the surface of the support.
The invention further relates to a method for preparing a molecularly imprinted polymer which comprises polymerising a composition comprising at least one monomer, and a template, on a support in a polymerisation medium with a free radical initiator, whereafter the template is removed from the molecularly imprinted polymer obtained, said polymerisation being confined to the surface of the support.
Still further the invention relates to the use of a molecularly imprinted polymer as defined above in chromatography, for separations, in chemical sensors, in molecular recognition as stationary phase in capillaries, in selective sample enrichment or in catalysis.
These and other advantages and characterising features of the present invention will appear from the following specification and the appended claims.